PROJECT SUMMARY DNA supercoiling is a ubiquitous feature of genomes, generated by the activity of translocating enzymes and by the binding of many proteins that wrap or change the twist of sequences to which they bind. Thus, genomic supercoiling is dynamic and is a sensitive regulator of genome-based activities, such as transcription and recombination. Most recent efforts have focused on transcription-generated supercoiling, the twin domain model, and the activity of topoisomerases. Largely unexplored is instead the impact of changes in DNA supercoiling on major aspects of cell physiology such as (i) long distance genomic interactions and (ii) the binding of architectural proteins to DNA. A satisfactory strategy to map genomic supercoiling (iii) is also lacking, since current approaches utilize probes that alter the structure of the double helix. Therefore, leveraging our expertise with magnetic tweezers, protein-mediated DNA looping, nucleoid associated proteins, and well established collaborations for in vitro and in vivo transcription assays, we have developed aims that will advance significantly our knowledge in these three fundamental areas: (1) Test the hypothesis that DNA supercoiling significantly facilitates the formation of topological structures, such as protein-mediated loops. Using the lac repressor protein (LacI) as a DNA looping protein, a range of loop lengths, and complementary in vitro and in vivo assays, we will establish the levels of supercoiling, tension and nucleoid associated proteins which most likely catalyze in vivo looping. (2) Test the hypothesis that DNA supercoiling affects the binding of proteins that define the architecture of the genome. We will establish the dependence on DNA topology of the (i) dissociation constant, and (ii) DNA compaction by representative, abundant nucleoid associated proteins (NAPs) that bind DNA non-specifically. (3) Test the hypothesis that promoter activity is affected, in a distance- and supercoiling-dependent manner, by an upstream protein-mediated loop.